Abstract
Rapid adaptations to various environmental changes are key to the survival of bacterial cells, which is mediated at the level of transcription initiation and RNA degradation/processing. Ribonuclease E (RNase E), an essential endoribonuclease gene in Mycobacterium species, is a key mediator in most reactions of RNA metabolism such as messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA) processing and their degradation qualifying it as an important drug target. Here, we first report the cloning of full-length Mycobacterium smegmatis mc2155 (M. smegmatis) RNase E [MSMEG_4626] and its expression with isopropyl β-D-1-thiogalactopyranoside (IPTG) in Escherichia coli (E. coli) as a glutathione S-transferase (GST) fusion protein. The best expression of the recombinant protein was optimized with respect to different parameters, such as E. coli strains, temperature, IPTG concentrations and time points of induction. Despite the use of protease inhibitors, we observed low-molecular-weight degradation products along with full-length fusion proteins. Maximum expression of soluble and least degraded full-length RNase E was obtained at 32 °C/0.1-mM IPTG, further purified by affinity chromatography using glutathione agarose beads and confirmed by immunoblotting technique using anti-GST antibody. In silico characterization using multiple sequence alignment, homology modeling and docking studies revealed four amino acid residues, Asp613, Phe657, Ile662 and Glu688, in the catalytic domain of RNase E that are completely conserved in both E. coli and Mycobacterium species, also involved in RNA binding and might act as candidate drug targets to combat antibiotic-resistant mycobacterial diseases. Additionally, recombinant GST-RNase E protein can unravel the mechanism of RNA degradation/processing through protein–protein interaction studies.
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Acknowledgements
LKJ, RKS and TN got the financial assistance from the Indian Council of Medical Research (ICMR) and University Grants Commission (UGC), New Delhi, respectively, as Senior Research Fellowship (SRF). We also acknowledge the central instrumentation facility of Interdisciplinary School of Life Sciences (ISLS), Banaras Hindu University, Varanasi, for providing us the instruments.
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L K J performed most of the experiments and mainly wrote the draft, R K S and T N helped in optimization of experimental conditions, V K S performed the bioinformatics study and A G designed the study and supervised the experiments and revised the final manuscript.
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From the known literature, it is concluded that RNase E is the central enzyme involved in all aspect of RNA metabolism. There is very little information known about mycobacterial RNase E structure and function. Therefore, authors selected this gene for their study. Further, its regulation and interactors can be studied to show its possible role as drug target for pathogenic mycobacterial diseases.
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Jaiswal, L.K., Singh, R.K., Nayak, T. et al. Heterologous Expression, Purification and Structural Characterization of Ribonuclease E from Mycobacterium smegmatis. Iran J Sci 47, 683–693 (2023). https://doi.org/10.1007/s40995-023-01467-x
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DOI: https://doi.org/10.1007/s40995-023-01467-x